?A- and ?B-crystallins are highly upregulated in the retina during diabetes; however, there is a fundamental gap in our knowledge regarding the role of this sustained increase on retinal cell death and vision loss. Such a gap limits our understanding and the potential use of ?-crystallins in developing novel therapies for the protection of retinal cells and prevention of vision loss associated with diabetes. The long-term goal of this research is to determine how the chaperone functions of ?-crystallins can be manipulated to preserve vision in patients with diabetes. The objective of this project is to identify mechanisms by which diabetes modulates the function of ?A- and ?B-crystallin proteins in retinal cells. The central hypothesis is that diabetes causes an initial adaptive upregulation o ?A- and ?B-crystallins that is neuroprotective, but over time specific post- translational modifications compromise this protective effect, leading to cell death and vision loss. This hypothesis is based on our published and preliminary data from several animal models of type 1 diabetes. The rationale for the proposed research is that identifying the function, regulation of expression, and post- translational modifications of ?-crystallins during diabetes will lead to strategies to enhance retinal cell viability in chronic retinal diseases such as diabetic retinopathy, uveitis, and age-related macular degeneration. This hypothesis will be tested by pursuing three specific aims: i) determine the role of ?A- and ?B-crystallins in retinal cell death and perturbations of visual functions during diabetes, ii) determine the effect of chronic diabetes on the regulation and function of ?-crystallin proteins, and iii) identify the mechanisms controlling ?A- and ?B-crystallin protective function in retinal neurons. Under the first aim, the progressive impact of diabetes on visual functions and retinal cell survival will be tested using our ongoing colonies of single and double ?A- and ?B-crystallin-knockout mice and approaches established and feasible in our laboratory. Under the second aim, combined proteomic and targeted mutation approaches will be used to identify the phosphorylation controlling ?-crystallins protective functions during diabetes. Under the third aim, biochemical, proteomic and cell biology methods will identify the mechanisms by which diabetes-associated cellular stresses regulate the protective function of ?-crystallins in retinal neurons. This research project is innovative because it involves studying the novel hypothesis that ?-crystallins protective function is progressively impaired during diabetes because of post-translational modifications and that it this function is a key element in retinal cell death and vision loss associated with diabetes. The proposed research is significant because identifying the role of potentially neuroprotective proteins such as ?-crystallins and the mechanisms by which they regulate visual function and retinal cell death during diabetes could lead to the development of new therapeutic strategies for diabetic retinopathy and other chronic retinal neurodegenerative diseases. PUBLIC HEALTH RELEVANCE: We will study how diabetes modulates the action of intrinsically protective proteins, called alpha- crystallins, in retinal cells. The goal is to understand how the natural adaptive mechanisms of protection in the retina are impaired by diabetes, leading to cell death and ultimately loss of vision. Understanding how the protective abilities of alpha-crystallins are affected during diabetes is relevant to the NIH's mission because it will enable us to develop novel approaches to manipulate these pathways. This would provide therapeutic benefit to preserve vision in persons with diabetes and potentially other retinal neurodegenerative disorders such as age- related macular degeneration.